Combination therapy method for treating chronic hepatitis B

ABSTRACT

The present invention is aimed at augmenting the success rate of using thymosin in treatment of chronic hepatitis B, by employing a combination therapy using thymosin with antiviral agents which are effective in inhibiting DNA synthesis or DNA polymerase during replication of the hepatitis B virus.

TECHNICAL FIELD

[0001] The instant invention relates to a method for treating chronichepatitis B infections. More specifically, the instant invention relatesto a method for treating chronic hepatitis B infections using boththymosin and an antiviral drug.

BACKGROUND OF THE INVENTION

[0002] Hepatitis B is the most prevalent form of hepatitis and is thesecond most common infectious disease worldwide. Worldwide estimates ofthose chronically infected with hepatitis B virus (HBV) are in excess of300,000,000. The disease is caused by the hepatitis B virus, a DNAvirus. The virus is transmitted through blood transfusions, contaminatedneedles, sexual contact and vertical transmission from mother to child.Moreover, a significant number of people are infected by unknown means.

[0003] Carriers of the virus can exhibit various forms of disease, oneof which is chronic hepatitis B. Approximately 50% of the carriers showchronic inflammatory changes in the liver and, of these, about 50% havehistopathologic changes, which are termed “chronic active hepatitis”,that may lead to fibrosis and ultimately to cirrhosis and progressiveliver failure. Carriers without chronic inflammatory changes may alsodevelop chronic active hepatitis. Liver cancer develops in about 10 to30% of hepatitis B carriers. It has been estimated that approximately 4million carriers of hepatitis B virus die each year from liver cancer orcirrhosis.

[0004] The persistent infection seen in individuals exhibiting chronichepatitis may be due to a defective or physiologically immatureimmunological response, resulting in an impaired ability to clear thevirus. Although the mechanisms responsible for liver damage are poorlyunderstood, it is thought that in most individuals such damage resultsfrom attack by the body's immune system on infected liver cells, ratherthan from liver destruction by HBV. Cytotoxic T cells appear to beresponsible for immune-mediated hepatic damage. The balance betweensuppression of immune system activity against normal tissue and theimmunological response mounted against the virus, also appears to beimpaired in chronic hepatitis B. A number of specific immune defectshave been described in chronic hepatitis, including defective productionof alpha-interferon by HBV-infected hepatocytes and inhibition ofcytotoxic T cell responses. (reviewed in Peters, M. et al., Hepatology(United States) (May 1991) 13(5):977-94).

[0005] Chronic infection is manifested by persistence of hepatitis Bsurface antigen (HB_(S)Ag) in the serum for more than 6 months. Thepresence of hepatitis B_(e) antigen (HB_(e)Ag) is associated with highlevels of viral replication.

[0006] The goals of treatment in chronic HBV infection include sustainedreduction of HBV replication (loss of HB_(e)Ag and HBV DNA), loss ofHB_(S)Ag, diminished infectivity, normalization of aminotransferaselevels, resolution of hepatic inflammation, improvement in symptoms, anda decreased rate of liver disease progression. The disappearance ofHB_(S)Ag from serum, implying termination of the HBV carrier state, hasbeen difficult to achieve with antiviral therapy.

[0007] Recent therapeutic trials have been directed towards utilizationof anti-viral agents, immunomodulators, immunosuppressives orcombinations of these. See, e.g. Thomas, H. C. et al., Seminars in LiverDisease (1986) 6(1): 34-41; Alexander, G. J. M. et al., Am. J. Med.(1988) 85: 143-146; Aach, R. D. et al., Ann. Int. Med. (1988) 109:89-91. At present, alpha-interferon is the only therapeutic approachthat has had regulatory approval in a number of countries. Interferon,however, induces a response in less than 50% of patients with chronichepatitis B, and has significant side effects that sometimes lead toearly cessation of therapy. Such side effects include fever, chills, andheadaches, followed by fatigue, anorexia, weight loss, emotionaldisturbances and in some cases, bone marrow suppression. (Perrillo, R.P. et al., Ann. Int. Med. (1988) 109: 95-100; Hoofnagle, J. H. et al.,Gastroenterology (1988) 95: 1318-1325. Other reported side effectsinclude rigors, alopecia, nausea and vomiting.

[0008] Various antiviral agents have been used as sole therapy agents inan attempt to treat chronic hepatitis B infection, including acyclovir,vidarabine, and adenine arabinoside. Sole therapy with these antiviralagents generally has been unsuccessful, either because the agent washighly toxic or resulted in some inhibition of viral replicationinitially, but failed to sustain viral replication inhibition long-term.See e.g. Alexander, G. J. M. et al., American J. Med. (1988), 85-2A:143-146.

[0009] Thymosin alpha-1 is a 28 amino acid peptide. The peptide,originally isolated from calf thymus thymosin fraction 5, is one ofseveral polypeptides present in thymosin fraction 5 which participate inthe regulation, differentiation and function of thymic dependentlymphocytes (T-cells). The isolation, characterization and use ofthymosin alpha-1 is described, for example, in U.S. Pat. No. 4,079,127.

[0010] Although the mechanism(s) by which thymosin alpha-1 mediates itseffects is unknown, evidence suggests that it may function throughmodulation of the immune system. Thymosin alpha-1 has been shown tocause increases in lymphocyte counts and enhance production ofgamma-interferon in individuals suffering from chronic active hepatitisB (Mutchnick, M. G. et al., Hepatology (1991) 14: 409-415).

[0011] Mutchnick, M. G. et al., “Thymosin: An Innovative Approach to theTreatment of Chronic Hepatitis B”, in Combination Therapies, Garaci, E.,Plenum Press, New York 1992, pps. 149-157, also describes the use ofthymosin alpha-1 in the treatment of chronic active hepatitis B, thatis, patients with evidence of liver injury upon biopsy. In 75% of thepatients in the study who received the peptide cleared hepatitis B virusDNA from serum and persistently tested negative for serum HBV DNA aftertreatment was terminated.

[0012] There remains an important need for a therapy for chronichepatitis B that efficiently and with fewer side effects attacks thevirus and modulates the immune response system.

SUMMARY OF THE INVENTION

[0013] The present invention is aimed at augmenting the success rate ofusing thymosin in treatment of chronic hepatitis B, by employing acombination therapy using thymosin with antiviral agents which areeffective in inhibiting DNA synthesis or DNA polymerase duringreplication of the hepatitis B virus.

[0014] The present invention provides a method for treating chronichepatitis B infections. In particular, the present invention relates toa method of treating subjects with chronic hepatitis B infection, byusing a combination chemotherapy regimen.

[0015] Accordingly, in one embodiment, the present invention is directedto a method for treating chronic hepatitis B infection in mammalscomprising administering to a subject having chronic hepatitis B atherapeutically effective amount of at least one thymosin, and aninhibitorily effective amount of at least one hepatitis B virusreplication or DNA polymerase inhibitor compound, either free or as apharmaceutically acceptable salt, in a pharmaceutically acceptablevehicle.

[0016] In a particularly preferred embodiment, the thymosin is thymosinalpha-1 and the hepatitis B virus replication or DNA polymeraseinhibitor compound comprises a nucleoside analog.

[0017] These and other embodiments of the present invention will becomeapparent by reference to the following description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a method for treating chronichepatitis B infection in mammals comprising concurrently administeringto chronic hepatitis B-infected subjects a therapeutically effectiveamount of at least one thymosin, and an inhibitorily effective amount ofat least one hepatitis B virus replication or DNA polymerase inhibitorcompound, either free or as a pharmaceutically acceptable salt, in apharmaceutically acceptable vehicle, which results in improved orbeneficially synergistic clinical effects in such subjects. Thesecombination therapies are more effective than when each is administeredas a sole treatment modality.

[0019] The term “thymosin” as used herein is intended to include anyimmunopotentiating polypeptide naturally occurring in the thymus glandor produced by chemical or recombinant means, or fragments derived fromany of these polypeptides. “Thymosin” includes, thymosin Fraction Five(TF-5), thymosin alpha-1 and any biologically active peptide fragment(such as C-terminal 4-28 and 15-28, and N-terminal 1-8, 1-14 and 1-20fragments), analog or derivative of any of those. As used herein, theterm “thymosin alpha-1” is intended to refer to the 28-mer describedbelow, with or without the N-acetyl group, as well as biologicallyactive analogs of the sequence (i.e. deletion, substitution and additionmutants), which are substantially homologous to the peptide sequenceshown below.

[0020] Thymosin Fraction Five (TF-5), originally described by Goldsteinet al. (Proc. Nat'l Acad. Sci. (USA), 69:1800-1803 (1972)), is apartially purified extract of bovine thymus containing at least 40peptide components, 20 of which have been purified to homogeneity ornear homogeneity; it contains about 0.6% of thymosin alpha-1. Low, etal., “Thymosins: Structure, Function and Therapeutic Application”,Thymus, 6:27-42 (1984), incorporated by reference.

[0021] A peptide that is “substantially homologous” to thymosin alpha-1is one in which at least about 30%, preferably at least about 85% toabout 90% and most preferably about 95%, of the amino acids match over adefined length of the molecule, with the sequence depicted below.

[0022] A “biologically active” fragment or analog of thymosin orthymosin alpha-1, is a fragment or analog of thymosin or thymosinalpha-1, respectively which retains a significant amount of the activityof the native molecule, i.e., which is capable of decreasing serum HBVDNA and/or hepatitis B surface antigen, as described further below.

[0023] The term “treatment” or “treating” as used herein refers toeither (i) the prevention of infection or reinfection (prophylaxis) or(ii) the reduction or elimination of indicators of chronic hepatitis B.

[0024] A “therapeutically effective amount” of thymosin is an amount ofthe peptide which has the capability of changing the measurableparameters of hepatitis B infection. The parameters that will normallybe monitored are serum hepatitis B surface antigen and serum viral DNA.Response is defined as a significant decrease of either of theseparameters. An amount of peptide which has the ability of eliciting aresponse is considered a “therapeutically effective amount.” HBV DNA canbe monitored using the spot hybridization assay described in Mutchnick,M. G. et al., Hepatology (1991) 14:409-415 and Lieberman, H. M. et al.,Hepatology (1983) 3:285-291, both of which are incorporated by referenceherein. Alternatively, the presence of HBV DNA in the blood can bemeasured using standard PCR technology. See, e.g. U.S. Pat. Nos.4,683,202 and 4,683,195, incorporated herein by reference in theirentirety. HBV DNA can also be detected through use of a commerciallyavailable kit from Abbott Laboratories, North Chicago, Ill. Serumhepatitis B surface antigen levels can be monitored using standard RIAs,as described by Mutchnick, M. G. et al., Hepatology (1991) 14: 409-415incorporated herein by reference, or by standard ELISAs.

[0025] The present invention relates to a combination therapy using amedicament containing as active ingredient at least one thymosin, eitherin free form or in the form of a pharmaceutically acceptable salt. Thethymosin may be administered alone or mixed with a pharmaceuticallyacceptable vehicle or excipient.

[0026] A most preferred embodiment of the present invention is to use amedicament containing as the thymosin active ingredient, thymosinalpha-1 for the treatment of chronic hepatitis B infection. The nativemolecule is a 28-mer, having the amino acid sequence shown below:

[0027] Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.

[0028] Thymosin alpha-1, as well as fragments and analogs thereof areeasily synthesized using standard methods of peptide synthesis, known tothose of skill in the art. U.S. Pat. Nos. 4,148,788 and 4,855,407describe the solution phase and solid phase synthesis, respectively, ofthymosin alpha-1, and are incorporated herein by reference in theirentirety. See also, Young, J. D., Solid Phase Peptide Synthesis, 2nd ed.(Pierce Chemical Company 1984); and Barany, G. and Merrifield, R. B.,The Peptides: Analysis, Synthesis, Biology, Vol. 2 (Gross, E. andMeienhofer, J. eds., Academic Press 1980), for a discussion of solidphase peptide synthesis; and Bodansky, M. Principles of PeptideSynthesis (Springer-Verlag 1984); and The Peptides: Analysis, Synthesis,Biology, Vol. 1 (Gross, E. and Meienhofer, J. eds., Academic Press1980), for solution phase peptide synthesis.

[0029] Thymosin alpha-1 can also be isolated directly from appropriatetissue expressing thymosin alpha-1, using techniques readily known inthe art. This is generally accomplished by first preparing a crudetissue extract which lacks cellular components and several extraneousproteins. The thymosin alpha-1 can be further purified, i.e. by columnchromatography, HPLC, immunoadsorbent techniques or other conventionalmethods well known in the art. U.S. Pat. No. 4,079,127 discloses amethod for purifying thymosin alpha-1 from calf thymus and isincorporated herein by reference in its entirety.

[0030] Thymosin alpha-1 and fragments or analogs thereof, can also beproduced recombinantly using methods well known to those of skill in theart. See, e.g. Sambrook, Fritsch & Maniatis, Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press 1989);Oligonucleotide Synthesis (M. J. Gait ed. 1984).

[0031] Thymosins can also be obtained from commercial sources (e.g.Alpha 1 Biomedicals, Inc., Foster City, Calif.).

[0032] Typically, the thymosin compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles. The active ingredient canbe mixed with vehicles containing excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitable vehiclesare, for example, water, saline, dextrose, glycerol, ethanol, or thelike and combinations thereof. In addition, if desired, the vehicle maycontain minor amounts of substances such as wetting or emulsifyingagents or pH buffering agents. Actual methods of preparing such dosageforms are known, or will be apparent to those skilled in the art. See,e.g. Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 15th edition, (1975). The composition or formulation to beadministered will, in any event, contain a quantity of the peptideadequate to reduce or eliminate HBV DNA and/or HBsAg from the serum ofthe subject being treated.

[0033] Thymosins may be administered orally or parenterally. Parenteraladministration may be achieved either intravenously, subcutaneously, orby intramuscular injection. Injectable formulations will contain aneffective amount of the active ingredient in a vehicle, the exact amountbeing readily determined by one skilled in the art. The activeingredient may range from about 1% to about 95% (w/w) of thecomposition, or even higher or lower if appropriate. The quantity to beadministered depends on factors such as the age, weight, health,severity of the condition, duration of treatment required of the subjectto be treated, and the other drugs of the combination of the inventionthat are being concurrently administered.

[0034] In the most preferred embodiment of the present formulations ofthe present invention, between about 300 μg to about 3000 μg, preferablybetween about 900 μg to about 1200 μg of thymosin alpha-1 per squaremeter of body area will be administered. Such dosages can be given oncea week up to once a day, preferably two to three times a week for atreatment course of between two months to three years, preferably forabout 6 to 12 months. A preferred dosage unit form for pharmaceuticaluse is 1.6 mg of lyophilized thymosin alpha-1 per vial, and thismaterial is reconstituted prior to use by the addition of diluent. Othereffective dosages can be readily established by one of ordinary skill inthe art without undue experimentation through routine dose responsetrials.

[0035] Additional formulations which are suitable for other modes ofadministration include suppositories and in some cases, aerosol,intranasal, oral formulations, and sustained release formulations. Forsuppositories, the vehicle composition will include traditional bindersand carriers, such as, polyalkaline glycols, or triglycerides. Suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%. Oral vehicles include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium, stearate, sodium saccharin cellulose, magnesiumcarbonate, and the like. These oral compositions may be taken in theform of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations, or powders, and contain from about 10% to about95% of the active ingredient, preferably about 25% to about 70%.

[0036] Intranasal formulations will usually include vehicles thatneither cause irritation to the nasal mucosa nor significantly disturbciliary function. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

[0037] Controlled or sustained release formulations are made byincorporating the peptide into carriers or vehicles such as liposomes,nonresorbable impermeable polymers such as ethylenevinyl acetatecopolymers and Hytrel® copolymers, swellable polymers such as hydrogels,or resorbable polymers such as collagen and certain polyacids orpolyesters such as those used to make resorbable sutures. The peptidescan also be presented using implanted mini-pumps, well known in the art.

[0038] Furthermore, the peptides may be formulated into compositions ineither neutral or salt forms. Pharmaceutically acceptable salts includethe acid addition salts (formed with the free amino groups of the activepeptides) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from freecarboxyl groups may also be derived from inorganic bases such as forexample, sodium, potassium ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0039] An inhibitorily effective amount of at least one antiviral agent,particularly hepatitis B virus replication or DNA polymerase inhibitorcompound is included in the combination chemotherapy regimen of thepresent invention. The infectious virion of hepatitis B contains asmall, circular DNA molecule that is partly single-stranded and a DNApolymerase that can make the DNA fully double-stranded. Its mechanism ofreplication involves an RNA intermediate. The antiviral agents which actto inhibit hepatitis B viral replication or inhibit DNA polymeraseactivity of the present invention comprise purine or pyrimidinenucleoside analogs.

[0040] An “inhibitorily effective amount” of an antiviral drug or agentis an amount of the drug which inhibits HBV virus replication, measuredby a decrease in viral DNA in the blood, as measured by PCR or othermethod known in the art.

[0041] Antiviral agents of the present invention which are pyrimidinenucleoside analogs include ddI, ddC, AZT and FIAU(fluoro-iodo-arabinofuranosyl-uracil) (see Table below). Antiviralagents of the present invention which are purine nucleoside analogsinclude acyclovir, ribavirin, ganciclovir, and vidarabine (see Tablebelow). AZT, ddC, ddI and FIAU act as polynucleotide chain terminators.Similarly, acyclovir and other purine analogs act as polynucleotidechain terminators. These analogs act as faulty substrates, thuspreventing DNA transcription. The mode of action of ribavirin is mostlikely interference with viral mRNA, resulting in inhibition of viralreplication.

[0042] The antiviral agents of the present invention, are given in anappropriate pharmaceutical dosage formulation. The pyrimidine nucleosideanalogs of the present invention can be given intravenously or orally tochronic hepatitis B-infected subjects at effective viral inhibitingdosages and according to regimens appropriate to the severity of thedisease and clinical factors. However, when given in combination with athymosin, a lower daily dosage for a subject can be devised according tothe clinical parameters and tests listed below. Those with skill in theart will, without undue experimentation, be able to devise dosagesdepending on the clinical condition of patients and the parametersdiscussed below.

[0043] Further, the thymosin and antiviral agent are administeredconcurrently in that the treatment administration of each drug overlapin time. Preferably, the antiviral agent is administered first with theadministration of thymosin beginning at the same time or within fourweeks after the first administration of the antiviral agent. Mostpreferably, administration of thymosin is begun within one week afteradministration of the antiviral agent has begun.

[0044] The following Table lists various antiviral agents of use in theinvention with exemplary modes of action and exemplary dosages and modesof administration. Antiviral Agents NAME CHEMICAL CLASS MODE OF ACTION¹TYPICAL DOSE² Zidovudine Pyrimidine analog Inhibits viral RNA- 200 mgq4h (AZT) dependent DNA polymerase (reverse transcriptase); chaintermination during DNA synthesis Acyclovir Purine analog •Inhibits DNA200 mg po q4h synthesis (DNA 5x/day for 10 polymerase) days •Blockschain elongation Topical IV 5-10 mg/kg q8h Ganciclovir Purine analog•Inhibits DNA IV 10 mg/kg per synthesis day •Inhibits DNA polymerase•Prevents chain elongation Vidarabine Purine analog •Inhibits DNA 15mg/kg/day IV polymerase •Prevents chain Ophthalmic elongation oint.Idoxuridine Pyrimidine analog Makes viral DNA more Ophth. oint.breakable Trifluridine Pyrimidine analog Inhibits DNA Ophth. soln.synthesis Foscarnet Inorganic Inhibits viral DNA IV 90-120 phosphonatepolymerase and mg/kg/day reverse transcriptase Amantadine Tricyclicamine Blocks assembly of 200 mg/day influenza virus Rimantadine Similarto Similar to 200-300 mg/day Amantadine Amantadine Ribavirin Purineanalog Multiple, including: •Inhibits synthesis Aerosol 1.4 of guaninemg/kg/hr nucleotides •Inhibits viral RNA 600-1800 mg/day polymerase po•Inhibits enzymes 4000 mg/day IV that cap mRNA Didanosine Purine analog•Blocks DNA chain 125-200 mg bid (dd1) elongation po •Competitivelyinhibits reverse transcriptase Zalcitabine Pyrimidine analog •Inhibitsviral DNA 0.75 mg q8h po (ddC) synthesis •Blocks DNA chain elongation•Inhibits reverse transcriptase FIAU

[0045] Antiviral agents are known and can be chemically synthesized orobtained commercially. For example: AZT, acyclovir and trifluridine(Burroughs Wellcome Co., Research Triangle Park, N.C.); ganciclovir(Syntex, Palo Alto, Calif.); vidarabine (Parke-Davis, Morris Plains,N.J.); idoxuridine (Smith Kline Beecham Pharmaceuticals, Philadelphia,Pa.); foscarnet (Astra Pharmaceuticals, Westborough, Mass.); amantadine(DuPont Pharmaceuticals (Wilmington, Del.)); rimantadine (ForestPharmaceuticals, Maryland Heights, Mo.); ribavirin (ICN Pharmaceuticals,Inc., Costa Mesa, Calif.); didanosine (Bristol Myers Squibb Company,Evansville, Ind.); zalcitabine (Roche Products, Nutley, N.J.).

[0046] In a preferred protocol, administration of a pyrimidine or purinenucleoside analog (e.g., AZT, ddI, ddC, FIAU, acyclovir, ribavirin) at adosage level and manner described in the Table is begun with thymosinalpha-1 at a dosage level of 1.6 mg. subcutaneously one to four timesweekly, most preferably twice weekly. Antiviral therapy is continueduntil viral DNA levels are negative and thymosin alpha-1 is continuedfor an additional three months.

[0047] Measurement of severity of the disease can be accomplished insubjects. Such measurements or markers of chronic hepatitis B includethe level of the enzymes ALT (alanine aminotransferase, sometimesreferred to as SGPT) and AST (aspartate aminotransferase, sometimesreferred to as SGOT) in the blood. These methods and techniques arestandard in this art.

[0048] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

EXAMPLE 1

[0049] A randomized, open trial with a total sample size of 20 patientsis conducted. Patients included in the study are at least 18 years ofage and are hepatitis B surface antigen seropositive for at least 6months. The patients also have HBV DNA in their serum on at least twooccasions at least one month apart (as measured by PCR or any otherstandard detection method). Only patients with chronic hepatitis B areincluded. These patients would have ALT levels at least 1½ times theupper limit of normal, and a liver biopsy showing changes consistentwith chronic hepatitis.

[0050] Patients which have had previous therapy with interferon withinone year, treatment with adrenocorticoid steroids within 6 months, HIVinfection as diagnosed by HIV seropositivity and confirmed by Westernblot, or the presence of hepatitis C virus antibody are excluded fromthe study, as well as patients who are pregnant or who have engaged inintravenous drug abuse within the previous 5 years from entering intothe trial, would be excluded.

[0051] Patients will undergo a pretreatment exam monthly for at leastone month prior to the treatment protocol. Examination includes:

[0052] 1. Blood studies including complete blood count (CBC) withdifferential count, platelet count, prothrombin time (PT), chemistrypanel including glucose, and creatinine, serum protein electrophoresis(SPEP), HB_(S)Ag, HB_(e)Ag and HBV DNA levels, alpha-fetoprotein (AFP),HB_(e)Ag antibody, HIV antibody, HCV antibody, and hepatitis-deltaantibody.

[0053] 2. Liver biopsy.

[0054] 3. Routine urinalysis.

[0055] 4. Women of child bearing age will have a pregnancy test.

[0056] Selected patients are randomized into two groups. Group 1 willreceive thymosin alpha-1 and one of the purine or pyrimidine nucleosideanalogs of the present invention (e.g., ddI, ddC, AZT, FIAU, ribavirin,acyclovir). Group 2 will receive a placebo. Group 1 patients are giventhymosin alpha-1 at a dosage of 1.6 mg/injection by the subcutaneousroute twice weekly and ddI at a dosage of 200 mg every 12 hours orallyfor 6 months. Group 2 patients receive placebo injections under the sameregimen. All patients are seen at 2 and 4 weeks from the start of thetreatment and thereafter at monthly intervals during the 6 monthtreatment period. They are also monitored monthly for 6 months to oneyear after completion of treatment.

[0057] Response to treatment is defined by loss of serum HBV DNA orserum HB_(S)Ag. A responder is a patient with decreased levels of serumHBV DNA or HB_(S)Ag achieved and sustained during the 12 month study. Anonresponder has no changes in either serum HBV DNA or HB_(S)Ag at theconclusion of the study (12 months). Relapse status is given to patientswho initially lose HBV DNA in their serum but regain the DNA by theconclusion of the study.

[0058] Patients are monitored for any significant side effects orallergic manifestations resulting from the treatment. The data will beanalyzed using a contingency table to compare responders andnonresponders in each group. The effect will be considered to besignificant if the percentage of responders in the combination therapygroup (group 1) is significantly higher than the percentage ofresponders in the placebo group (group 2), with a p value < 0.05.

EXAMPLE 2

[0059] This protocol is similar to example 1, except the antiviral drugddI will be given until HBV DNA levels are negative, and thymosinalpha-1 will be given for three months beyond that time.

EXAMPLE 3

[0060] These protocols are similar to examples 1 and 2, except theanti-viral drug is ddC given in a dose of 0.75 mg orally every 8 hours.

EXAMPLE 4

[0061] These protocols are similar to examples 1 and 2, except theanti-viral drug is AZT given in a dose of 200 mg orally every 4 hours.

EXAMPLE 5

[0062] These protocols are similar to examples 1 and 2, except theanti-viral drug is ribavirin given in a dose of approximately 200 mgorally every 6 hours.

[0063] Thus, methods of treating chronic hepatitis B infection aredisclosed. Although preferred embodiments of the subject invention havebeen described in some detail, it is understood that obvious variationscan be made without departing from the spirit and scope of the inventionas defined by the appended claims.

What is claimed is:
 1. A method for treating chronic hepatitis Binfection in mammals comprising concurrently administering to a subjecthaving chronic hepatitis B a therapeutically effective amount of atleast one thymosin, and an inhibitorily effective amount of at least onehepatitis B virus replication or DNA polymerase inhibitor compound,either free or as a pharmaceutically acceptable salt, in apharmaceutically acceptable vehicle.
 2. The method of claim 1 whereinsaid thymosin is thymosin alpha-1.
 3. The method of claim 1 wherein saidhepatitis B virus replication or DNA polymerase inhibitor comprises apyrimidine nucleoside analog.
 4. The method of claim 1 wherein saidhepatitis B virus replication or DNA polymerase inhibitor comprises apurine nucleoside analog.
 5. The method of claim 1 wherein saidadministering is done subcutaneously.
 6. The method of claim 1 whereinsaid thymosin is thymosin alpha-1 and said hepatitis B virus replicationor DNA polymerase inhibitor is selected from the group consisting ofpurine nucleoside analogs and pyrimidine nucleoside analogs.
 7. Themethod of claim 1 wherein said thymosin is thymosin alpha-1 and saidhepatitis B virus replication or DNA polymerase inhibitor is ddI.
 8. Themethod of claim 1 wherein said thymosin is thymosin alpha-1 and saidhepatitis B virus replication or DNA polymerase inhibitor is ddC.
 9. Themethod of claim 1 wherein said thymosin is thymosin alpha-1 and saidhepatitis B virus replication or DNA polymerase inhibitor is ribavirin.10. The method of claim 1 wherein said thymosin is thymosin alpha-1 andsaid hepatitis B virus replication or DNA polymerase inhibitor is AZT.11. The method of claim 1 wherein said thymosin is thymosin alpha-1 andsaid hepatitis B virus replication or DNA polymerase inhibitor isacyclovir.
 12. The method of claim 1 wherein said thymosin is thymosinalpha-1 and said hepatitis B virus replication or DNA polymeraseinhibitor is FIAU.
 13. The method of claim 3 wherein said analog isselected from the group consisting of ddI, ddC, AZT, and FIAU.
 14. Themethod of claim 4 wherein said analog is selected from the groupconsisting of acyclovir, didanosine, ribavirin, ganciclovir, andvidarabine.
 15. The method of claim 1 wherein from about 300 μg to about3000 μg of said thymosin per square meter of body area is administered.16. The method of claim 1 wherein said thymosin is administered twiceweekly.
 17. The method of claim 15 wherein said thymosin is administeredsubcutaneously twice weekly and ddI is administered in a regimen ofabout 200 mg orally twice a day.
 18. The method of claim 15 wherein saidthymosin is administered subcutaneously twice weekly and ddC isadministered in a regimen of about 0.75 mg orally three times a day. 19.The method of claim 15 wherein said thymosin is administeredsubcutaneously twice weekly and ribavirin is administered in a regimenof about 200 mg orally four times a day.
 20. The method of claim 15wherein said thymosin is alpha-1 and is administered subcutaneouslytwice weekly and AZT is administered in a regimen of 200 mg orally sixtimes a day.
 21. The method of claim 15 wherein said thymosin is alpha-1and is administered subcutaneously twice weekly and said acyclovir isadministered in a regimen of 200 mg orally six times a day.